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. 2015 Aug 21;11(8):e1005381. doi: 10.1371/journal.pgen.1005381

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© 2015 Deng et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Fig 1 — A-D. Dia colocalizes with the actin focus at the site of fusion. A. Fusing myoblast (FCM, false colored magenta in all Figures) and FC/Myotube (false colored turquoise in all Figures, see Methods and S1 Fig) in a stage 15 twist-actin::GFP embryo stained for F-actin (phalloidin, white) and antibodies against Dia (red) and GFP (green). Dia accumulates at the fusion site, colocalizing with the F-actin focus. B. Signal intensity plot confirms Dia enrichment with actin at the fusion site. Average fluorescence intensity measured across the F-actin foci as shown in A. A line of predetermined length was dropped across the fusion site; fluorescence intensity along the line was measured in different channels, normalized, and plotted (n = 10). See Methods and S2 Fig for details on intensity measurements and normalization. C. Fluorescent intensity curves with error bars for both control (n = 10) and proteins of interest (n = 10). D. Still images from a time-lapse series of a fusion event in a stage 14 embryo expressing Dia::GFP and Moesin::mCherry driven by DMef2-Gal4 indicates that Dia::GFP has the same spatial and temporal pattern as actin at the fusion site. Moesin::mCherry (red) labels F-actin at the fusion site (arrows). Di. Signal intensity curve showing Dia::GFP (green) and Moesin::mCherry (red) colocalize during fusion at each time point. E-H. Dia is localized to the fusion site in both the FC/Myotube (turquoise) and FCM (magenta). Dia::GFP (green), phalloidin (white), endogenous Dia (red) E. duf5.1-Gal4 driven Dia::GFP shows expression in myotubes/FCs in stage 14 embryos. Fusing FCM and myotube were captured before cytoplasmic mixing. Dia antibody staining (red) is present at the F-actin focus (phalloidin, white). FC driven Dia::GFP (green) expressed in FC/Myotubes partially overlaps with endogenous Dia at the fusion site. F. The signal intensity curve confirms that the peak of FC driven Dia::GFP partially overlaps with endogenous Dia. G. Stage 16 sns-Gal4 driven Dia::GFP shows expression in mbc ^C1 mutant FCMs, where fusion is blocked. Staining as in F. Dia enrichment is seen on FCM side with both Dia (red) and FCM driven Dia::GFP (green). H. Signal intensity curve confirms Dia::GFP and Dia overlap and are within the F-actin peak. Scale bar: 2.5μM